玉米SRAP反应体系的建立与优化

SRAP标记(Sequence-related Amplified Polymorphism,序列相关扩增多态性)是一种新的分子标记技术,具有简便、稳定、中等产率、在基因组中分布均匀的特点,是开展遗传图谱构建、基因定位与克隆、比较基因组学c、DNA指纹图谱、生物多样性、预测杂种优势等研究的一个很实用的工具。对玉米SRAP反应体系的程序、模板、Mg2+等参数进行优化研究,结果表明玉米的PCR程序为:94℃预变性5 min,94℃变性1 min,35℃复性1 min,72℃延伸1 min,5个循环;94℃变性1 min,50℃复性1 min,72℃延伸1 min,35个循环,最后72℃延伸10min;25μl反应体系中,模板量为20 ng,Mg2+浓度为2.0 mmo1/L。该研究建立的玉米SRAP体系重复性好,稳定性强。

Optimization of SRAP System in Maize

Sequence-related amplified Polymorphism(SRAP) is a molecular marker technology newly developed by Li and Quiros,which has the characteristics of simplicity,reliability,moderate throughput ratio and distributed evenly in genome.It has been widely applied in the fields of genetic map construction,plant comparative genomics,gene mapping and cloning,cDNA fingerprinting and genetic diversity.In order to adopt SRAP approach in the study on maize germplasm and heterosis,optimizing experiments for the SRAP PCR reactions including PCR Protocol,concentrations of DNA template and Mg~(2+) were carried out.The results showed that the most suitable Protocol was initially denaturing at 94℃ for 5 min;then 94 ℃,1 min;35 ℃,l min and 72 ℃,1 min for the first five cycles,then the annealing temperature was raised to 50 ℃ for another 35 cycles.The optimum concentrations of DNA template and Mg~(2+) were 20 ng DNA/25 μ1 and 2.0 mmol/L Mg~(2+) in this SRAP-PCR system.The new establishment of SRAP system of maize was fully reproducible and with good stability.